The present invention relates generally to heat- or thermo-stable enzymes and, more specifically, to enzymes that have been protected by a non-reducing saccharide, for example, trehalose, to improve their heat stability during feed processing. The enzyme compositions have an increased stability during steam treatment due to the presence of the saccharide and can be used in animal feed or food composition that are steam treated.
The potential commercial application of enzymes for maximizing animal performance through the improvement of nutrient digestion is significant. Unfortunately, the main obstacle for the full development of this market is the observation that a variety of enzymes are intrinsically unstable. This instability becomes apparent in two main areas. First, enzymes may lose activity during storage before use. This issue can be partially managed by strict control of the storage conditions. More significant is the loss of activity during the various processing conditions in animal feed production. Certainly under pelleting conditions many enzymes are inactivated. Although feed pelleting represents a cost in terms of energy, it provides several benefits. It is well known that pelleting increases the digestibility of the starch fraction. Besides the higher bioavailability of some nutrients there is also less feed waste, a more uniform nutrition and improved feed handling because of reduced dustiness. Pelleting also becomes more important in the context of food safety. Most microorganisms are sensitive to heat under conditions of high moisture. Therefore the feed industry increasingly uses steam pelleting in order to reduce the microbiological load of the feed.
Pelleting also becomes more important in the context of food safety. Feed crises during recent years have had enormous consequences causing a decrease of consumer confidence in the feed industry. Today, consumers only want safe food products. The modern feed industry must now establish procedures and practices in order to manage the risks associated with their activity. The major hazards in feed can only be controlled by strict application of HACCP and GMP as critical and basic food safety management systems.
There has been much emphasis upon Salmonella control in recent years and it is now well recognized that raw materials and feeds can be readily contaminated by Salmonella. Another concern with Salmonella today is the prevalence of strains with multiple antibiotic resistances. In recent years, both in Europe and the United States, the presence of Salmonella typhimurium DT 104 has been reported, initially in cattle, but now spreading to other species and this serotype has multiple resistance to many antibiotics. Control of Salmonella in pig production to reduce Salmonella contamination in pig carcasses is also an important food safety concern.
Programs for the control of Salmonella and other pathogens in raw materials and feeds usually require either heat processing or chemical treatment with bacterial inhibitors. A combination of heat and chemical processing is also sometimes used, particularly in manufacturing critical feeds such as those for high-value poultry breeding stock.
Salmonella are sensitive to heat under conditions of high moisture and an example is pasteurizing milk where heating at 70° C. or above for 15 seconds is sufficient to destroy Salmonella. However, in the animal production industries it is important to appreciate that raw materials and animal feeds are basically dry materials with a moisture content usually below 14-15%. Salmonella are resistant to drying and become more heat resistant as they dehydrate. Salmonella may survive on surfaces and in dust for years. Davie and Wray (Persistence of Salmonella Enteritidis in poultry units and poultry feed, British Poultry Science 37, 589-596 (1996)) showed that S. Enteritidis and other serotypes persisted in poultry feed for at least ten months. Therefore feed production increasingly makes use of steam pelleting in order to reduce the microbiological load of the feed.
The supplementation of diets with enzymes in order to improve the digestibility of animal feeds is an established practice since more than a decade, Unfortunately the harsh conditions that destroy microorganisms are also detrimental to enzymes. The increasing application of heat treatment of feeds poses a significant challenge for the development and use of enzyme feed supplements. Enzymes are complex protein structures that obtain their typical activity from their amino acid composition together with their three-dimensional structure. It is generally known that most enzymes are susceptible to heat treatment processes. In the pelleting process, feed is first subjected to a conditioner phase to prepare the feed for being pressed into pellets. In the conditioner, direct stream addition takes place and the average moisture content of feed increases. Steam addition also results in an increase of the temperature up to 60-95° C. The combination of a high environmental temperature and the presence of water for a certain time period cause enzyme denaturation. The enzymes will often irreversibly loose their conformation and consequently also their enzymatic activity. A solution to this problem, which has been applied for many years, is to apply enzymes after the pelleting process by spraying enzymes in the form of liquid solutions onto the pellets after they are cooled.
The improvement of heat stability is an important challenge in the context of enzyme development for the animal feed industry. It is known that enzymes can be stabilized by encapsulation in a protecting matrix. The loss of enzyme activity may be overcome by “immobilization” or “encapsulation” of enzymes (Faber, K. Biotransformations in Organic Chemistry, 4th ed.; Springer, Berlin (2000)). These techniques involve either the attachment of an enzyme to a solid support (coupling onto a carrier) or linkage of the enzyme molecules to each other (cross-linking). Alternatively the biocatalyst may be confined to a restricted area from which it cannot leave but where it remains catalytically active (entrapment into a solid matrix or a membrane restricted compartment). As a consequence, homogeneous catalysis using a native enzyme often turns into heterogeneous catalysis when immobilized enzymes are employed.
The use of immobilized enzymes in animal feed adds another level of complexity to this subject matter. Strict heterogeneous catalysis would imply that the enzyme is not soluble anymore and cannot be separated from a feed sample with simple extraction techniques. This phenomenon will pose serious difficulties for the recovery of the enzyme activity in the feed. It will be impossible to measure enzyme activities, which is a strict requirement for registration. It is possible to circumvent this issue by using a water-soluble matrix. Optimally this matrix will remain intact during the pelleting, but should dissolve easily in the digestive tract in order to liberate the enzymes (Gray, C. J. Thermostability of Enzymes, ed: M. N. Gupta, Springer, Berlin (1993)).